> Peptide Synthesis Services


Synthetic peptides are synthesized using a Rainin Symphony Multiplex 12 column synthesizer, an Liberty Microwave Peptide Synthesizer and an Aapptec 396 Multiple Organic Synthesizer. Currently these machines allow for synthesis scales of 20 µmoles, 25 µmoles and multiples of 100 µmoles and can yield peptides in 10's of mgs to 100's of mgs, depending on the peptide length. Due to the inherent individual characteristics of each peptide, many times certain sequences will offer challenges to their successful synthesis. Therefore, it is suggested that investigators conservatively estimate the amount of peptide required (in mgs) for their study and allow the Macromolecular Synthesis staff to review the relative difficulty of the synthesis and then determine the scale that most likely will produce the desired amount.

Standard Peptide Synthesis

The standard synthesis is usually done on a polystyrene resin appropriate for the specific peptide sequence being requested. The smallest scale syntheses (<50 mg) are usually run on the ACT 396. Those syntheses requiring more than 50 mg or that require online monitoring due to problem sequences or special chemistries are run on the Rainin Symphony. Standard syntheses are cleaved from their resins yielding a free acid form of the peptide having a negatively charged C-term and a positively charged N-term. Peptides greater than 40 amino acids in length have been synthesized that passed our QC requirements. [It should be noted that peptides longer than 20-25 residues generally will require extra synthesis time due to the inherent difficulties that the longer sequences present]

Modifications to Standard Peptide Synthesis

  • 96-Well Pin Synthesis: A 96-well format manual synthesis of peptides containing diketopiperizine C-termini or peptides covalently bound to polystyrene pins is available. [See related methods]
  • Peptide Amides and Acetylated Peptides: C-terminus amides and N-terminus acetylations can be incorporated during synthesis. [See related methods]
  • Multi Antigenic Peptides: 4-branch and 8-branch synthesis available for enhanced antibody production. [Contact staff for limitations]
  • Phosphorylated Amino Acids: Phosphoserine, Phosphothreonine and Phosphotyrosine can be incorporated during synthesis.
  • Amino Hexanoic Acid Linker: Alkane 6 carbon linker can be added to either termini or internally during synthesis.
  • Biotinylation: N terminal biotinylation is available during synthesis. Select biotinylated amino acids such as Lysine can be incorporated internally during synthesis. [See related methods]
  • Fluorescent Labeling: Currently, FITC/Amino Hexanoic Acid complex, fluorescein, FAM, Dansyl Chloride or Mca can be added to the N terminus during synthesis.

Post-Synthesis Modifications

  • KLH Coupling: Coupling of synthetic peptides to KLH for antibody production is available.
  • Sepharose Coupling: Coupling of synthetic peptides to a succinylhexanoic linker attached to 4B sepharose beads for affinity column production is available.
  • Global Reactions: Acetylations, Biotinylations and Dye Labelings can be done globally post cleavage.

PNA Synthesis

The Macromolecular synthesis has recently started offering Peptide Nuclecic Acid (PNA) synthesis as well as peptide-PNA conjugate synthesis. [Contact staff for details]

Peptoid Synthesis

The Macromolecular synthesis has recently started offering Peptoid synthesis. [Contact staff for details]

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How to Order

You may want to check out our section on Related Methods before ordering for the first time.

  1. Select the amount needed - Calculate the amount needed to complete your study.
  2. Select or briefly describe how the peptide will be used - Describe how the peptide will be used in the study so allowances can be made during the synthesis based on the end use. Also, indicate any bioactivity the peptide may have.
  3. Enter the name of the peptide - Enter a name for your peptide such that you will be able to distinguish it from other peptide synthesis.
  4. Enter the sequence - Enter the desired sequence using the one letter abbreviation in the order form table. Be sure to note the choices for phosphorylated amino acids. If "X" or "Z" is used in the sequence, please completely explain what is to be used in that position.
  5. Select synthesis modifications - Select any synthesis modifications required such as N-term biotinylation, fluorescence labeling, acetylation or C-term amide.
  6. Select post-synthesis needs - Select KLH or Sepharose coupling if needed

Order consultation and conformation

It is the goal of the Macromolecular Synthesis staff to produce the highest quality products available for St Jude’s investigators. It is highly recommended that when ever possible the researchers utilize the expertise of the staff. Sometimes delays can be avoided and cleaner peptides obtained by small sequence modifications. Also, based on the amount required, the relative difficulty of the synthesis, the intended use of the peptide and any post synthesis requirements the staff will suggest the appropriate scale and level of purity needed to satisfy the needs of the request. After consulting and recommending changes if necessary, an order conformation will be sent to the person initiating the order.

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Quality Assurance

Two quality control methods are currently used to monitor post-peptide syntheses. The Waters Alliance High Performance Liquid Chromatography (HPLC) is used to determine the purity of each peptide. (25µmole and greater). Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometry is also used to evaluate all peptides, see proteomics.

While Fmoc peptide synthesis is a proven synthesis protocol, there are times when improper assembly or post-synthesis difficulties causes unforeseeable problems. Avoiding or modifying difficult sequences can minimize these. (see related methods below). When this is not possible, most synthesis problems are characterized by the rigorous quality control employed, documented and reported to the requestor when synthesis is completed. If the peptide does not give the anticipated results, a re-evaluation of the sequence or a review of the synthesis and/or post-synthesis handling may be necessary. If the activity of the peptide changes over time, it may be necessary for the user to request additional QC work done to verify that the peptide has not gone under a post-synthesis modification.

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Instrumentation

Peptides are synthesized using a Rainin Symphony Multiplex Organic Synthesizer, an Liberty Microwave Peptide Synthesizer, and an Aapptec 396 Multiple Organic Synthesizer. The combination of these machines allows for the synthesis from 10 mg to several hundreds of mgs of peptide.  The Rainin Symphony Multiplex Organic Synthesizer is able to run 12 independent syntheses concurrently while arrays containing as many as 96 peptides can be synthesized simultaneously on the Advanced ChemTech 396 in smaller quantities.  The Liberty is the first peptide synthesizer to use microwave energy to increase the rate of peptide coupling reactions.  The Liberty is configured to make one peptide at a time, with the capibility for programmed automated, consecutive synthesis of multiple peptides.  The graetest impact of the Odyssey is our ability to fill small orders in a timely fashion, reducing waiting times to 2-3 days in some cases.

Purifiaction

HPLC purification of peptides is done upon request.  A Waters Alliance HPLC system and a Beckman BioSys 500 system are used for HPLC purification.

Fees

Please follow this link to a general fees page.

ConsiderationS Related to the purity and solubility of synthetic peptides

Investigators should bear in mind the following considerations related to the purity of chemically synthesized peptides, and the conditions required to utilize peptides in experiments.

Water - The Major Non-Peptide Contaminant

The most abundant substance likely to be present in preparations of chemically synthesized peptides, other than the desired peptide product, is water.   Peptides are typically rendered as "fluffy" lyophilized powders that are strongly hygroscopic, rapidly absorbing water from the air.   The peptide content of a weighed amount of powder may be measured in absolute terms by amino acid analysis to an accuracy of ±10%.   Powders average 70% peptide by weight.

Investigators may request amino acid analysis for accurate quantitation, which is performed by contract laboratories, or may perform spectrophotometric quantitation based on calculated extinction coefficients. (Details available upon request).

Minor Non-Peptide Contaminants

Other minor non-peptide impurities may also be present.   These include residual acid and thiol scavenger reagents derived from the cleavage/deprotection reaction that is performed at the end of the synthesis. These unwanted materials are typically present at very low levels because all peptides are washed extensively in diethyl ether to remove them.   However, occasionally a peptide may have an associated odor signaling the presence of residual thiol compounds, or may yield a solution of low pH if dissolved at high concentration in unbuffered water.   These contaminants are usually unimportant if peptides are used at low concentrations, e.g. µmol or nmol.   However, if peptides are employed at mmol concentrations, as may be the case in NMR studies, low level non-peptide contaminants may affect the system.

In such cases, investigators should request the Hartwell Center to desalt peptides using reverse-phase trap cartridges or some other suitable method.

Peptide Contaminants

The most abundant peptide-related contaminants, when present, are peptides that retain side-chain protecting groups at low stoichiometry. Investigators should be aware that cases may arise in which peptide analogs of these kinds may interact with target molecules at much higher affinity or speed than the desired peptide product.   In unfavorable cases, such contaminants may be present at levels well below the range detectable by the chromatographic or mass spectrometric quality control procedures performed routinely on all synthetic peptides, yet dominate the biochemical behavior of the peptide preparation.   The likelihood of such artifactual results may be minimized by performing high resolution chromatographic fractionation of peptides, e.g. by reverse-phase HPLC. Because side-chain protecting groups are typically large, hydrophobic moieties, peptides retaining them may often be separated from the desired product.

Chromatographic purification may be requested at additional cost, although the process could add several days to the production process, the exact time depending upon how much peptide needs to be purified.

Peptide Solubility

Oftentimes, peptides may be readily dissolved in aqueous solvents at high concentrations.   However, sometimes solubilization is a challenge.   The following guidelines may be utilized to solubilize peptides in difficult cases: Where the peptide contains several residues with ionizable side chains (Asp, Glu, His, Lys, Arg), dissolve the peptide initially at extreme pH (pH 1-2 in the case of peptides with strongly basic pI, or pH 10-11 for peptides with strongly acidic pI), then neutralize by titration with the aid of pH indicator paper or pH-sensitive electrode.   Note that the side-chains of phosphoserine and phosphothreonine residues are unstable at high pH and should not be exposed to pH values above 8.0.

Where the peptide contains predominantly non-polar and uncharged polar residues, dissolve initially in a solvent containing a mixture of water and organic solvent.   Methanol, acetonitrile, isopropanol, and dimethylsulfoxide are increasingly strong solvents for hydrophobic peptides.   Dilute the solubilized peptide in water, but bear in mind the possibility that strongly hydrophobic peptides may re-precipitate when the dielectric constant of the solvent rises.

Although peptides may be dissolved in chaotropes such as urea or guanidine hydrochloride, such reagents are rarely compatible with biochemical assays.   Remember never to heat a peptide in the presence of urea, because this will assuredly result in carbamylation of amino groups.

The Special Challenge of Cysteine-Containing Peptides

At neutral and alkaline pH, cysteine-containing peptides will undergo oxidation of their thiol groups with intra- or inter-chain disulfide bridge formation.   This normally creates molecular species unintended in the design of all but the most carefully planned experiments.

Disulfide formation can be prevented by maintaining the pH of peptide solutions = 5.0.   Alternatively, cyteine residues may be alkylated to yield carbamideomethyl or carboxymethyl derivatives, or oxidized to cysteic acid.   These modifications may be performed by Hartwell Center staff if requested.    To avoid thiol-related trouble, however, investigators may wish to consider replacing cysteine residues by serine where the substitution is allowable.

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Related Methods

Storage and Handling of Synthetic PEPTIDES

Peptides are delivered lyophilized in a closed container. Long-term storage of peptides in solution is not recommended. While some peptides can be stored at room temperature, storage in a 20° C dessicator is recommended. When peptides are removed from cold storage, they should be allowed to warm to room temperature. Whatever method is used, storage should minimize moisture content. Over time, all peptides are susceptible to various modifications such as cysteinyl oxidation, hydrolysis of labile amide bond in glutamine and asparagine residues and at C-termini and cleavage at aspartic residues to name a few. Therefore, it is recommended that peptides stored for long periods of time (even lyophilized) be reanalyzed before use to check for sequence integrity.

Pin Synthesis - Synthesis of Peptide Arrays

The Hartwell Center provides small scale synthesis of peptide arrays (Mimotopes). These peptide arrays, also available from Chiron, can now be synthesized by the Hartwell Center at substantially less cost. Mimotopes are available in several different forms, for different applications. Some of the applications of mimotopes and the advantages of the different types of mimotopes are described below

Uses of Peptide Arrays:

B-cell assays
Arrays of peptides permanently bound to polyethylene pins can be used for screening binding sites in standard ELISA procedures or for epitope mapping. Since the peptides remain attached to the pins, the array of peptides can be used repeatedly to screen with many different antibodies.

T-cell Assays
Peptide arrays can be produced which can be cleaved under sterile conditions directly into tissue culture media. The peptides are well suited for use in assays of T-cell proliferation induced by the peptides.

Protein Scanning
Location of binding sites of antibodies or other protein-binding molecules can be accomplished by synthesis of overlapping peptides covering the length of the protein and assessing the ability of each region of the protein to bind the antibody. In order to cover the entire length of the protein, a large number of peptides must be synthesized. For the larger scale synthesis methods available from the Hartwell Center, this would require an unreasonable amount of time and would be very expensive. Only a small amount of each peptide is required to perform the binding assay. Once the region to which the molecule binds has been located other arrays can be synthesized to further characterize the region of interest.

Binding Site Localization
Once the binding region has been located, the specific sequence responsible for binding can be studied. To locate the minimum peptide sequence required for binding, a second mimotope is synthesized. This assay would contain a series of peptides of different lengths corresponding to the binding region. Assessment of the ability of these peptides to bind provides precise localization of the region of the protein responsible for binding.

Interaction of specific amino acids on binding
The contribution of each amino acid in the binding region can be investigated by producing an array of peptides corresponding to the binding region but each having one amino acid replaced in the sequence, usually by alanine. The contribution of the side chain of each amino acid in the binding region can be investigated.

Effect of Mutations at Specific Sites
Mimotopes can also be produced which represent peptides corresponding to a region of interest, but each containing a different amino acid at a specific position. The potential effect of mutating a specific site can be investigated.

Effect of Phosphorylation
Peptide arrays containing phosphorylated amino acids can be also be produced. The effect of phosphorylation can be investigated in conjunction with the methods described above by synthesis of arrays of both phosphorylated and non-phosphorylated versions of the peptides.

Biotinylated Peptide arrays
Arrays of peptides containing an N-terminal biotin can be produced. Isolation of the peptides, and any molecules bound to them, from the solution is possible by binding of the peptides to avidin.

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Available Array types:

C-terminal Amide and Free acid
These peptides correspond in structure to the peptides available on larger scales from the Hartwell Center. The arrays are synthesized at a scale of 5 µmoles, typically yielding 3-5 mg of peptide. They are provided as lyophylized powders in individually labelled tubes and can be used in various assay types. Once the peptide of interest is identified, the same peptide can be made on a larger scale for further investigations. Free acid and amide peptides will be checked by MALDI-TOF Mass Spectrometry to insure that the correct peptide was produced.

DKP Peptides
Peptides containing a cyclic Lys-Pro dipeptide (DKP) on their C-terminus are available in a 1 µmole scale. The DKP group has been shown to have no effect on binding in many applications. The peptides that are supplied are attached to pins in a holder that fits into a standard 96-well microtiter plate. The peptides can be cleaved from the pins directly into a sterile, neutral pH solution, which is useful for cell culture based assays. The deprotected peptides are supplied still attached to the pins along with a procedure for sterilizing the pins and removing the peptides. They will contain 2 control peptides which are used to assure a successful synthesis. No quality control procedures will be done by the Hartwell Center on these peptides since they are not removed from the pins.

Noncleavable Pins
Peptides attached to polystyrene pins through a noncleavable linker on their C-terminus are available in a 1 µmole scale. The peptides that are supplied are attached to the pins in a holder that fits into a standard 96-well microtiter plate. The peptides can be used directly in 96-well format assays such as ELISA without removing them from the pins. The holder is designed to hold the pin containing each peptide suspended in a different well of a microtiter plate. They will be supplied, ready for use, attached to a 96 pin block that will fit into a standard microtiter plate. They will contain both positive and negative control peptides which can be used in conjunction with a control antibody (available from Chiron) as an assay control. No quality control procedures will be done on these peptides since they are not removed from the pins.

Peptide Design Considerations

This synthesis method uses the same conditions for the synthesis of all of the peptides in the mimotope since they are all synthesized at the same time. The synthesis conditions cannot be optimized for each peptide, possibly resulting in lower purity and yield for some of the peptides in the set. The proper design of the peptides and inclusion of appropriate controls is essential for this method to provide reliable data. For mimotopes where a protein or a region of a protein sequence is being scanned, the peptides should be designed to overlap one another in the protein sequence. If enough overlap exists in the peptides, low yield of one peptide can be compensated since the binding site on that peptide also exists in the overlapping region of one or more other peptides. In experiments where a specific amino acid is important, such as replacement of amino acids by alanine or studying the effects of site-specific mutations, at least two of each peptide sequence should be synthesized and included in the assay. Use of duplicate peptides can also act as controls for nonspecific binding. In addition, the native sequence should be included as a positive control and in some cases an unrelated sequence can be included as a negative control.

DKP and noncleavable mimotopes are supplied still attached to the pins. For these peptide types, suitable control peptides are essential since no quality control can be performed on these peptides. Standard ELISA positive and negative control pins are included in these arrays. These can be assayed using a standard control antibody available from Chiron. When possible, the requested peptides should also include other controls as appropriate to the particular assay procedure being performed.

How to Request Mimotopes

Two methods are available for generating peptides.

  1. You can send a protein sequence to the Hartwell Center from which the peptides can be generated or
  2. You can generate the peptides yourself and submit a request for the desired sequences.

A computer program designed by Chiron to aid in generating suitable peptides from a protein sequence is available in the Hartwell Center. The sequence must be in GCG format and can either be retrieved from one of the protein databases or entered using the GCG SEQED program. Contact the Macromolecular Synthesis Staff to arrange for assistance in using this program. If the sequence is in a database, bring the accession number and name of the sequence to the Hartwell Center. If the sequence has been typed in SEQED by the investigator or was obtained from a source other than one of the available databases, bring the sequence as a text file on a floppy disk to the Hartwell Center. Once the peptides have been generated by this program, you will receive an electronic request form by E-mail. This form will contain the generated peptides for editing or approval. The remaining portions of the request form must then be completed according to the instructions provided in the form. The completed request form should be sent by E-mail back to the Hartwell Center to complete the request.

If desired, you may design your own peptides. An electronic request form is available from the Hartwell Center which will allow the investigator to type the peptides manually in the proper format. The desired peptide sequences should be entered and the additional information provided, per the instructions, in the request form. The completed request form should be sent to the Hartwell Center via E-mail.

Click to view PIN References

Peptide Amides and Acetylated Peptides

C-terminal amidation and N-terminal acetylation are available options on peptides synthesized by the Macromolecular Synthesis Section. One obvious application for these blocked peptides is to mimic naturally occurring blocked peptides. The N-termini of many proteins are naturally blocked and acetylation can be used to similarly block synthetic peptides. Some peptides, especially peptide hormones, are naturally amidated at their carboxyl-terminus and synthesis of amidated peptides can be used to mimic these naturally occurring peptides.

There is another application for these types of blocked peptides which is not so obvious. These blocking groups can be used to shield charges resulting when a peptide is removed from the interior of a protein.

As an example, imagine that you want to synthesize a peptide corresponding to the sequence in the interior of the protein highlighted below.

When this peptide is removed from the surrounding protein, at neutral pH, the amino and carboxyl termini each exist as charged species, with the amino terminus being positively charged and the carboxyl terminus being negatively charged. If this peptide is intended to mimic an internal piece of the protein, the ends of the peptide possess charges, which were not present, when the peptide was within the protein.

When the ends of the peptide are blocked, by carboxylation at the amino-terminus and amidation of the carboxyl-terminus, they remain uncharged, better resembling the condition in the protein.

One consideration before using this method to block the charges on the peptide is that the blocking groups should not interfere with the application planned for the peptide. If the peptide were to be conjugated by the amino terminus to KLH for antibody production, amino terminal acetylation would prevent the coupling. Similarly, a carboxyl terminal amide may interfere with applications involving the carboxyl terminus. Provided the blocking groups do not affect the experiment, amidation and acetylation of the termini of a peptide are an effective method of removing the charged ends of the peptide. Other blocking groups, such as biotin and fluorescein, are available for other applications, if needed.

Selection of Sequences for Synthetic Peptides

Success in the synthesis of peptides depends greatly on the sequence chosen. While the main determinant of the choice of a sequence is the requirement of the application, there is often some flexibility in which segment of a protein sequence is chosen for synthesis. To the extent permitted by the application, selection of a sequence that avoids problematic regions will improve the quality of the resulting peptide. Some general rules for identifying sequences, which are difficult to synthesize, are presented below.

Problematic Sequences at the Amino Terminus

  1. Avoid glutamine and glutamic acid at the N-terminus. During cleavage of the peptide from the synthesis resin and upon subsequent storage, glutamine and to a lesser degree glutamic acid can cyclize to form pyroglutamate.

Problematic Sequences in the Interior of the Peptide

  1. Select a peptide containing as few arginines as possible. Arginine is a difficult amino acid to couple and a difficult amino acid from which to remove the side chain protecting groups. This results in peptides with deletion of the arginine and peptides that possess protected arginine derivatives.
  2. Avoid sequences where aspartic acid is followed by glycine, serine, asparagine or threonine. In this context aspartic acid can cyclize to form a succinate or aspartamide. Cleavage of the cyclic derivative results in isomerization of the aspartic acid to both the D and L forms as well as formation of both D and L- b-aspartic acid.
  3. Avoid sequences that contain several of the same amino acid in a row. Appearance of several of the same amino acid in sequence can form unique packing structures, which can create difficulty in coupling because the N-terminus can become inaccessible.
  4. Avoid peptides containing cysteine or methionine. Cysteine and methionine can oxidize during synthesis or during storage.

Problematic Sequences at the Carboxyl Terminus

  1. Avoid proline at the C-terminal position or the second position from the C-terminus. Proline in combination with several amino acids can form a cyclic diketopiperazine at the dipeptide stage of the synthesis. Once this cyclization occurs, synthesis can proceed no further.
  2. Avoid histidine at the C-terminus. Histidine is subject to isomerization, especially when coupled to the synthesis resin. This isomerization results in a mixture of two peptides with L-histidine and one with D-histidine at the C-terminus.

These simple rules do not cover all of the difficult sequences for peptide synthesis but can help in avoiding those problem sequences, which are easily identified. Often one or more of the problem sequences described above cannot be avoided. All of these problems can be overcome during synthesis, but the more of these difficulties which must be dealt with during synthesis, the less likely the synthesis will be successful. By considering the difficulty of the peptide when selecting the sequence for synthesis (to the extent allowed by the application) you can improve the likelihood of obtaining good synthetic peptides.

Sepharose-Immobilized Peptides

Synthetic peptides can be attached to a sepharose resin for use in affinity chromatography. The peptide is typically attached to the resin by a short hydrocarbon spacer in order to avoid steric effects from the resin. The peptide can be attached by either the amino- or carboxyl-terminus. There are several applications for these immobilized peptides. When making antibodies, binding the peptide used for the initial immunization to sepharose can enhance the specificity of antibodies to the peptide. Most non-specific proteins and antibodies will not bind to the peptide and are washed away while the desired antibody remains bound to the resin. The enriched antibody is then released from the resin, usually by washing the resin with ammonium carbonate solutions at high pH. (Please contact the Hartwell Staff for current protocols)

In addition to antibodies, other proteins that bind to a particular peptide sequence can be isolated by binding to an immobilized form of the peptide. In this application, the bound protein can be removed from the resin after washing away the nonbinding proteins either by changes in pH or salt concentration or by competition with the unbound form of the peptide. One common use for this procedure is the isolation of proteins that bind to a peptide sequence when it is phosphorylated, but not to the unphosphorylated peptide. Two resins are needed for this method, one containing the unphosphorylated peptide and the other containing the phosphorylated peptide. A mixture of proteins is added to the resin containing the unphosphorylated peptide. The solution containing the proteins that do not bind to this resin are then applied to the resin containing the phosphorylated peptide. After washing away the proteins that do not bind to this resin, the proteins that specifically bind the phosphorylated peptide sequence are released and analyzed.

Proteins can also be attached to sepharose for use in identifying peptides, proteins or oligonucleotides that bind to the immobilized protein. Attachment of peptides and proteins to sepharose is available in the Center for Biotechnology. Attachment to other supports is also possible.

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PIN References

Carson, J., Patel, D., Maylor, J., Lunney, O., Shepherd, P.S., Best, J.M. and McCance, D.J. (1989) Identification of Immunological Regions of the major coat protein of human papilloma virus type 16 that contain type-restricted epitopes, J. Gen. Virol., 70, 2973.

Conlon, J.W., Clarke, I.N. and Ward, M.E. (1988) Epitope mapping with solid-phase peptides: Identification of type subspecies- and genus-reactive antibody binding domains on the major outer membrane of Chlamydia trachomatis. Mol Microbiol., 2, 673.

Das, M.K. and Lindstrom, J. (1991) Epitope mapping of antibodies to acetylcholine receptor a subunits using peptides synthesized on polypropylene pegs. Biochemistry, 30, 2470-2477.

Goudsmit, J., Boucher, C.A., Meloen, R.H., Epstein, L.G., Smit, L.A., Van der Hoek, L. and Bakker, M. (1988) Human antibody response to a strain specific HIV-I gp120 epitope associated with cell fusion inhibition. J. Acquired Immune Deficiency Syndromes, 2, 157-164.

Kazemi, M. and Finkelstein, R .A.(1991) Mapping epitopic regions of cholera toxin B-subunit protein. Mol. Immunol., 28, 865-876.

Novak, J., Sova, P., Krchnak, V., Hamsikova, E., Zavadova, H. and Roubal, J. (1991) Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides. J. Gen. Virol., 72, 1409-1413.

Papsidero, L.D., Sheu, M. and Ruscetti, F.W. (1989) HIV type 1-neutralizing Mabs which react with P17 core protein: characterization and epitope mapping. J. Virol. 63, 267-272.

Ralston, S., Hoeprich, P.D., and Akita, R. (1989) Identification and synthesis of the epitope for a human monoclonal antibody which can neutralize human T-cell leukemia/lymphotropic virus type I. J. Biol. Chem., 264, 16343.

Snijders, A., Benaissa-Trouw, B.J., Oosteriaketi, T.A.M., Puijk, W.C., Posthtimus, W.P.A., Meloeti, R.H., Baere, W.A.M., OosLing, J.D., Kraaijeveld, C.A. and Snippe, H., (1991) Identification of linear epilopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine. J. Gen. Virol., 72, 557-565.

Tan, X., Ratnam, M., Huang, S., Smith, P.L. and Freisheim, J.H. (1990) Mapping the antigenic epitopes of human dihydrofolate reductase by systematic synthesis of peptides on solid supports. J. Biol. Chem., 265, 8022-8026.

Van der Zee, R., van Eden, W., Meloen, R.H., Noordzij, A. and van Embden, J.D.A. (1989) Epitope mapping and characterization of a T-cell epitope by the simultaneous synthesis of multiple peptides. Eur. J. Immun., 19, 43-47.

Wang, J-X., DiPasquale, A.J., Bray, A.M., Maeji, N.J. and Geysen, H.M. (1993) Study of Stereo-requirements of substance P binding to NK1 receptors using analogues with systematic D-amino acid replacements. Bioorganic & Medicinal Chemistry Letters, 3, 451-456.

Burrows, S.R., Rodda, S.J., Suhrbier, A., Geyesn, H.M.. and Moss, D.J. (1992) The specificity of recognition of a cytotoxic T lymphocyte epitope. Eur. J. Immunol. 22, 191-195.

Corradin, G., Etlinger, H.M. and Chiller, J.M. (1977) Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice. J. Immunol. 119, 1048.

Demotz, S., Matricardi, P., Lanzacecchia, A. and Corradin, G. (1989) A novel and simple procedure for determining T cell epitopes in protein antigens. J. Immunol. Methods. 122, 67.

Gammon, G., Geysen, H.M., Apple, R.J., Pickett, E., Palmer, M., Ametani, A. and Sercarz, E.E. (1991) T-cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes. J. Exp. Med. 173, 609-617.

Suhrbier, A., Rodda, S.J., Ho, P.C., Csurhes, P., Dunckley, H., Saul, A., Geysen, H.M. and Rzepczyk, C.M. (1991) Role of single amino acids in a T-cell epitope from tetanus toxin. J. Immunol., 147, 2507-2513.

Van Oers, M.G.J., Pinkster, J. and Zeijlemaker, W.P. (1978) Quantification of antigen-reactive cells among human T Lymphocytes. Eur. J. Immunol., 8, 477.

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